Processing at the Beginning, Middle, and 3 End of mRNAs

1
Processing at the Beginning, Middle, and (3-)
End of mRNAs
1. Capping at the 5-end
2. Polyadenylation at the 3-end
3. Editing in the middle (internal sequences)
2
Early Probing of Cap Structure
First caps characterized in viral RNAs (1975)
Fig. 15.1
RNA labeled with 3H from AdoMet and 32P from
labeled nucleotides
DEAE-cellulose after KOH treatment
Further work ?-phosphoryl group is present in
the cap, suggesting that the cap is at the 5-end
Blocking group at the 5 -end shown to be m7G
3
Cap Structure
7-methylguanosine
m7G linked 5-5 through triphosphate
The number of 2-O-methyl nucleotides can vary
from 0 to 2. Cellular RNAs usually have 1 or 2.
Triphosphate linker
2-O-methyl nucleotide
Fig. 15.3
4
Cap Synthesis
pp
Four reaction steps
Fig. 15.4
5
The Cap Is Added Early During Transcription
Guanylyltransferase structure
Triphosphatase
Guanylyltransferase
methyltransferase
Gu and Lima, Curr. Opin. Struct. Biol. 15 (2005)
99-106
6
Four Functions of 5 Cap Structure
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
7
Four Functions of 5 Cap Structure
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
Reovirus RNAs synthesized in vitro
Capped, blocked (GpppG), or uncapped RNA
After 8 h in Xenopus oocytes
Fractionated over glycerol gradient
Fig. 15.6
8
Four Functions of 5 Cap Structure
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
9
Four Functions of 5 Cap Structure
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
Cap facilitates transport out of the nucleus
Mattaj and colleagues placed an RNA that is
normally transcribed by RNA polymerase II and
capped (U1 snRNA) under control of RNA polymerase
III. It was not capped and was retained in the
nucleus.
10
Four Functions of 5 Cap Structure
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
11
Modification of the 3-end Polyadenylation
Darnell and colleagues digested HeLa cell RNA
with RNases A and T1, cleaving after C, U, and G
(but not As)
Isolated by electrophoresis poly A of 150200 nt
Fig. 15.7
12
Four Functions of Polyadenylation
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
13
Four Functions of Polyadenylation
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
Globin RNA injected into oocytes
Rate of translation measured
Fig. 15.9
14
Four Functions of Polyadenylation
1. Protection from nucleases
2. Enhancement of translation
3. Roles in transport
4. Enhancement of splicing
Effect of poly(A) and cap tested in rabbit
reticulocyte lysate
Here, no difference was observed in stability
(possibly because of different system?)
We will save the mechanism of translation
activation by poly(A) until we study translation
Fig. 15.10
15
Mechanism of Polyadenylation
Fig. 15.12
16
A Sequence Element Determines the Polyadenylation
Site
In mammals, the sequence AATAAA is 20 bp
upstream from polyadenylation site
Fig. 15.15
17
A Sequence Element Determines the Polyadenylation
Site
This recognition sequence is followed about 20
bp downstream by a GU-rich sequence
Yeast genes usually lack an AAUAAA sequence and
it is difficult to identify a consensus beyond
being AU-rich
Plants have AAUAAA but are more tolerant of
substitutions than animals
Fig. 15.16
18
The Process of Polyadenylation
First, the pre-mRNA is cleaved downstream from
the AAUAAA signal
CPSF (Cleavage and polyadenylation specificity
factor) binds to the AAUAAA sequence, and CstF
binds to the G/U region. Both conclusions are
based on crosslinking results.
CF I and CF II (cleavage factors) are also
required. Poly A polymerase (PAP) is probably
also present, because polymerization rapidly
follows cleavage
RNA polymerase is also involved, because its
presence enhances activity
Fig. 15.19
19
The Process of Polyadenylation
Then the cleaved pre-mRNA is extended with
poly(A) in two phases
Wickens and colleagues used purified poly(A)
polymerase and CSPF to extend a synthetic RNA
Fig. 15.20
Fig. 15.25
20
The Process of Polyadenylation
Then the cleaved pre-mRNA is extended with
poly(A) in two phases
Demonstration of two phases
Labeled RNAs added to HeLa nuclear extract
Minimum of A10 needed to overcome AAGAAA
Fig. 15.25
Fig. 15.21
21
The Process of Polyadenylation
Poly(A)-binding protein II (PAB II) also
enhances elongation
AAGAAA
Fig. 15.25
Fig. 15.24
22
The Process of Polyadenylation
23
Poly A Is Shortened Over Time In the Cytoplasm
HeLa cells RNA labeled for 48 hrs with 3H and 32P
Nuclear and cytoplasmic RNA isolated and analyzed
by gel electrophoresis
Fig. 15.26
24
mRNAs Can Also Be Edited After Transcription
Unusual editing events discovered in
mitochondria of trypanosomes
Fig. 16.14
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mRNAs Can Also Be Edited After Transcription
Fig. 16.15
26
Editing Uses Guide RNAs
Editing proceeds from 3 to 5 using guide RNAs
Fig. 16.18
Fig. 16.17
27
Mechanism of Editing
Fig. 16.20
28
Editing By Adenosine Deamination (ADAR Enzymes)
3 ADARs in humans
A to I changes Q codon to R codon
One protein affected is a brain Ca2 channel,
GluR
Reenan, Trends in Genetics, 17 (2001), 53-56
29
Key Points
1. Eukaryotic mRNAs have a defined structure at
the 5-end called a cap. The cap serves several
functions, including protection of the mRNA from
degradation and enhancement of translation.
2. Eukaryotic mRNAs are also processed at the
3-end by addition of a poly A sequence. This
addition serves many of the same purposes as the
cap.
3. RNAs can also be edited internally. In this
process the sequence is changed so that the
sequence of the protein does not correspond to
the DNA sequence.
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